Drosophila Melanogaster Mariya V Zhukova, Elena Kiseleva*

Zhukova and Kiseleva BMC Microbiology 2012, 12(Suppl 1):S15 http://www.biomedcentral.com/1471-2180/12/S1/S15 RESEARCH Open Access The virulent Wolbachia strain wMelPop increases the frequency of apoptosis in the female germline cells of Drosophila melanogaster Mariya V Zhukova, Elena Kiseleva* Abstract Background: Wolbachia are bacterial endosymbionts of many arthropod species in which they manipulate reproductive functions. The distribution of these bacteria in the Drosophila ovarian cells at different stages of oogenesis has been amply described. The pathways along which Wolbachia influences Drosophila oogenesis have been, so far, little studied. It is known that Wolbachia are abundant in the somatic stem cell niche of the Drosophila germarium. A checkpoint, where programmed cell death, or apoptosis, can occur, is located in region 2a/2b of the germarium, which comprises niche cells. Here we address the question whether or not the presence of Wolbachia in germarium cells can affect the frequency of cyst apoptosis in the checkpoint. Results: Our current fluorescent microscopic observations showed that the wMel and wMelPop strains had different effects on female germline cells of D. melanogaster. The Wolbachia strain wMel did not affect the frequency of apoptosis in cells of the germarium. The presence of the Wolbachia strain wMelPop in the D. melanogasterw1118 ovaries increased the number of germaria where cells underwent apoptosis in the checkpoint. Based on the appearance in the electron microscope, there was no difference in morphological features of apoptotic cystocytes between Wolbachia-infected and uninfected flies. Bacteria with normal ultrastructure and large numbers of degenerating bacteria were found in the dying cyst cells. Conclusions: Our current study demonstrated that the Wolbachia strain wMelPop affects the egg chamber formation in the D. melanogaster ovaries. This led to an increase in the number of germaria containing apoptotic cells. It is suggested that Wolbachia can adversely interfere either with the cystocyte differentiation into the oocyte or with the division of somatic stem cells giving rise to follicle cells and, as a consequence, to improper ratio of germline cells to follicle cells and, ultimately, to apoptosis of cysts. There was no similar adverse effect in D. melanogaster Canton S infected with the Wolbachia strain wMel. This was taken to mean that the observed increase in frequency of apoptosis was not the general effect of Wolbachia on germline cells of D. melanogaster,it was rather induced by the virulent Wolbachia strain wMelPop. Background cells remain undamaged [3,4]. Apoptosis is a complex Apoptosis, a form of programmed cell death, is a pro- process whereby a proteolytic cascade of caspases is cess needed for normal development and maintenance activated in cells [5]. of tissue homeostasis in multicellular organisms [1,2]. The occurrence of apoptosis is a feature of female Cells undergoing apoptosis show characteristic changes, germline development common to vertebrate and inver- such as chromatin and cytoplasm condensation, chro- tebrate species [6,7]. In the Drosophila melanogaster mosomal DNA fragmentation, breaking up of nuclei and ovaries, there are two checkpoints where programmed then of cells into fragments called apoptotic bodies cell death occurs. One is in the germarium (region 2a/ [3,4]. Individual cells apoptose, while the neighboring 2b), where apoptosis probably regulates the proper ratio of germline cells to follicle cells [8]. The other check- * Correspondence: [email protected] point is located in the vitellarium (stages 7-8 of oogen- Institute of Cytology and Genetics, Siberian Branch of the Russian Academy esis) [9]. The number of egg chambers undergoing of Sciences, Novosibirsk, 630090 Russia © 2012 Zhukova and Kiseleva; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Zhukova and Kiseleva BMC Microbiology 2012, 12(Suppl 1):S15 Page 2 of 12 http://www.biomedcentral.com/1471-2180/12/S1/S15 apoptosis increased in D. melanogaster fed a diet lacking from D. melanogaster Canton S. In contrast, the number protein [8], under the effect of 900-MHz and 1800-MHz of germaria containing apoptotic cells in the checkpoint radiation [10], and after exposure to chemical agents was considerably increased in the wMelPop-infected [11]. The normal development of mature egg is consis- flies as compared with their uninfected counterparts. tently associated with apoptosisof15nursecellsinthe Thus, evidence was obtained indicating that the virulent egg chamber [12]. It is noteworthy that apoptosis and Wolbachia strain wMelPop has an effect on the fate of autophagy coexist at all the above mentioned stages of germline cells during D. melanogaster oogenesis. oogenesis in D. melanogaster [13,14]. It has been also hypothesized that the apoptotic pro- Results cess had a symbiotic origin [15]. In terms of the endo- Frequency of apoptosis in germaria from ovaries of the symbiotic theory, mitochondria, which play a major role uninfected and Wolbachia-infected D. melanogaster at the early stages of apoptosis, evolved from the free- Two parts are distinguished in the Drosophila ovariole: living prokaryotes [5]. One of the symbionts may be the germarium made up of four regions (1, 2a, 2b, 3) involved in the regulation of apoptosis in partner cells. and the vitellarium (Figure 1A, B) [27,28]. The region To illustrate, extracellular parasites, particularly such 2a/2b, where apoptosis can occur, contains 16-cell cysts, worms as filarial nematodes, schistosomes and the ces- somatic stem cells (SSCs), which contact with the tode Taenia crassiceps, are able to induce apoptosis in somatic stem cell niche (SSCN) and follicle cells (Figure host immune cells [16]. Bacterial pathogens (Chlamydia, 1B). Cell death in this region of the germarium was Neisseria, Legionella pneumophila) can either block or detected by two methods, acridine orange (AO)-staining induce apoptosis in host cells, depending on the stage of and TUNEL assay. Fluorescence microscopy of AO- infection [17,18]. At the early stage of infection, bacteria stained ovarioles demonstrated that apoptotic cells were replicate in the host cell, using different mechanisms to located as large yellow or orange spots in region 2a/2b prevent apoptosis. At the late stages of infection, the of the germarium from D. melanogaster (Figure 2A, C, bacteria induce apoptosis in the host cell, thereby facili- E, G). Germaria containing no apoptotic cells fluoresced tating egress and ensuring infection of neighboring cells. homogeneous green (Figure 2B, D, F, H). It should be Wolbachia associated with various hosts in which it noted that wMel- and wMelPop-infected flies, besides manipulates viability and reproduction causing parthe- bright spots in region 2a/2b (Figure 2C, G), showed nogenesis, feminization, male killing and cytoplasmic weak punctuate fluorescence both in regions 2a/2b and incompatibility, provides a unique model for studying 1ofthegermarium(Figure2C,D,G,H).Suchfluores- mechanisms of symbiont interactions [19,20]. The Wol- cent puncta were not observed following TUNEL, bachia strain wMel is widely spread in natural popula- thereby indicated that they were not caused by tions of D. melanogaster [21,22]; in contrast, wMelPop apoptosis. has been detected in a laboratory stock of D. melanoga- The percentage of germaria containing apoptotic cells ster [23]. It is possibly not encountered in nature. In D. was 41.8±4.1% in the uninfected D. melanogasterw1118T melanogaster, the wMelPop strain reduces lifespan, pro- maintained on standard food, whereas it increased to liferating widely in the brain, muscle and retina cells 70.6±5.3% in the wMelPop-infected flies (Figure 2I). [23]. In certain insect species, the presence of Wolba- Analysis performed with the wMel-infected D. melano- chia is required for oogenesis [24]. Removal of the Wol- gaster Canton S revealed no significant differences from bachia strain wAtab3 from parasitic wasps Asobara their uninfected counterparts (Figure 2I, Table 1).The tabida resulted in copious apoptosis of the egg cham- next step was to exclude the possible effect of insuffi- bers in the ovarioles and led to sterility [25]. The cient nutrition on the current results. To do so, we con- mechanisms whereby the endosymbiont Wolbachia ducted experiments in which flies were raised on rich impacts apoptosis in host cells have been poorly studied. food source taking into account that it decreases the Preferential infection and high accumulation of Wolba- number of germaria containing apoptotic cells [8,29]. chia in region 2a of the germarium [26] where the We found that rich food causes a decrease in the rela- checkpoint is located in Drosophila was thought-provok- tive proportion of apoptotic germaria in both w1118T ing. We raised the question: Can bacteria Wolbachia in and w1118 flies; however, the difference between these region 2a of the germarium affect the frequency of two groups was significant (Figure 2I, Table 1). The per- apoptosis there? Using fluorescence and transmission centage of germaria containing apoptotic cells did not electron microscopy we compared germaria from ovaries change under the effect of rearing D. melanogaster Can- of two D. melanogaster stocks infected

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